Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays

Chantal B.F. Vogels; Anderson F. Brito; Anne Louise Wyllie; Joseph R Fauver; Isabel M. Ott; Chaney C. Kalinich; Mary E. Petrone; Marie-Louise Landry; Ellen F. Foxman; Nathan D. Grubaugh

By testing each of the nine primer-probe sets using 10-fold dilutions of SARS-CoV-2 RNA derived from cell culture ( Fig. 2A ) or 10-fold dilutions of SARS-CoV-2 RNA spiked into RNA extracted from pooled nasopharyngeal swabs taken from patients in 2017 (SARS-CoV-2 RNA-spiked mocks; Fig. 2B ), we again found that the PCR amplification efficiencies were near or above 90% ( Fig. 2C ). To measure the analytical sensitivity of virus detection, we used the cycle threshold (Ct) value in which the expected linear dilution series would cross the y-intercept when tested with 1 genome equivalent per μL of RNA. Our measured sensitivities (y-intercept Ct values) were similar among most of the primer-probe sets, except for the RdRp-SARSr (Charité) set ( Fig. 2D ). We found that the Ct values from the RdRp-SARSr set were usually 6-10 Cts higher (lower virus detection) than the other primer-probe sets. qRT-PCR assays. We compared nine primer-probe sets and a human control primer-probe set targeting the human RNase P gene with 10-fold dilutions of ( A ) full-length SARS-CoV-2 RNA and ( B ) pre-COVID-19 mock samples spiked with known concentrations of SARS-CoV-2 RNA. We determined ( C ) efficiency and ( D ) y-intercept Ct values (measured analytical sensitivity) of the nine primer-probe sets. We extracted nucleic acid from SARS-CoV-2-negative nasopharyngeal swabs (collected from respiratory disease patients in 2017) and spiked these with known concentrations of SARS-CoV-2 RNA. Symbols depict sample types: squares represent tests with SARS-CoV-2 RNA and diamonds represent SARS-CoV-2 RNA-spiked mock samples. Colors depict the nine tested primer-probe sets. The CDC human RNase P (RP) assay was included as an extraction control.